TMC GENES We have generated mice segregating knockout (null) alleles of Tmc1 and Tmc2. We are characterizing their mutant auditory and vestibular (balance) phenotypes. Mice that are homozygous for the Tmc1 knockout allele are deaf. Mice that are homozygous for the Tmc2 knockout allele have normal hearing and balance. Mice that are homozygous for knockout alleles of both genes are deaf and have abnormal vestibular function. These results indicate that both Tmc1 and Tmc2 are required for normal vestibular function (balance), whereas only Tmc1 is required for hearing. We are collaborating with Dr. Charley Della Santina who has evaluated vestibular function in the mice to demonstrate that their abnormal balance and gait is due to abnormal function of the semicircular canals (cristae) otolithic organs of the vestibular system. We are currently working to identify the intracellular location and function of TMC1 and TMC2 proteins in hair cells. We used a yeast two-hybrid screen to isolate genes encoding proteins that potentially interact with TMC1. We narrowed the list to a few candidate genes implicated in vesicular trafficking and in antiapoptosis and cell survival. We are using a combination of approaches to determine which interactions occur in situ in hair cells. We generated knockout mice for Tmc6 and Tmc8 to better understand the function(s) of Tmc genes and proteins. Mutations in human TMC6 or TMC8 genes cause epidermodysplasia verruciformis, a recessive disease resulting in chronic cutaneous HPV infections (papillomas) with increased susceptibility to non-melanoma skin cancers. We have done extensive RNA expression analyses to show that Tmc6 and Tmc8 are primarily expressed in lymphoid cells and tissues and lung and skin, and primarily during development. The homozygous knockout mice have no obvious phenotypic abnormalities, so we are collaborating with Dr. Paul Lambert to determine if these mice have alterations in their susceptibility or response to papillomavirus infection. TWIRLER MOUSE We have identified a candidate mutation and generated a knock-in mouse line with this mutation to confirm its pathogenicity. The resulting line has all of the phenotypic features of Twirler, thus proving the pathogenic role of the candidate mutation. ENLARGED VESTIBULAR AQUEDUCTS (EVA) We are ascertaining families with multiple members with nonsyndromic EVA that is not associated with detectable SLC26A4 mutations or Pendred syndrome. We are using those families in a genetic linkage-based strategy to identify other genetic causes of EVA. We are evaluating several candidate regions of potential linkage. We generated a doxycycline-inducible Slc26a4-expression mouse line. This transgenic mouse line allows us to manipulate Slc26a4 expression (on an Slc26a4-knockout background) by the administration of doxycycline in drinking water. We have defined a time window during which Slc26a4 is required for auditory development and function. We can manipulate doxycycline administration to generate mice in which there is significant residual hearing and isolated EVA, a phenotype which models human EVA more closely than the existing knockout mouse. Based upon results from other laboraties demonstrating that wild type Slc26a4 contributes to the pathogenesis of asthma and hypertension in mice, we postulated that SLC26A4 mutations may confer a protective effect against the development of hypertension or asthma in humans. We analyzed medical records for our cohort of EVA probands and their family members to address this question. None of 21 individuals with two mutant alleles of SLC26A4 had hypertension or asthma, but this observation was not statistically significant. A larger study is required to test this important hypothesis. We collaborated with Drs. Sheikh Riazuddin and Thomas Friedman to complete a genotypic survey for deafness caused by SLC26A4 mutations in Pakistan. Eight novel mutations were identified and the study showed that SLC26A4 mutations account for approximately 7.2% of severe to profound, prelingual-onset deafness in Pakistan. SLC26A4 mutations are thus the most common known cause of deafness in that population. We collaborated with Dr. Byung Choi of the NIDCD and Dr. Seung-Ha Oh of Seoul National University to define the prevalence and spectrum of SLC26A4 mutations among a cohort of Korean patients with EVA. Based upon those results, we developed a hierarchical mutation screening strategy involving denaturing high performance liquid chromatography and direct sequencing. We validated this strategy in silico with published east Asian EVA cohorts to show that it is an efficient and cost-effective strategy for the molecular genetic diagnosis of EVA in east Asian populations. COLLABORATIVE PROJECTS We collaborated with Dr. Thomas Friedman of the NIDCD to complete a genotypic survey for DFNB9 deafness caused by OTOF mutations in Pakistan. We identified 10 novel mutations and showed that OTOF mutations account for approximately 2.3% of severe to profound, prelingual-onset deafness in Pakistan. We collaborated with Dr. Thomas Friedman of the NIDCD to map the autosomal dominant deafness locus DFNA27 to chromosome 4q12-13.1. We collaborated with Dr. Thomas Friedman of the NIDCD to map the autosomal recessive deafness locus DFNB79 to chromosome 9q34.3. We collaborated with Dr. Thomas Friedman of the NIDCD to identify an autosomal recessive deafness gene as LRTOMT, a fusion gene with alternative reading frames. We collaborated with Drs. Leslie Biesecker of the NHGRI and Matthew Kelley of the NIDCD to identify low-frequency sensorineural hearing loss as a novel and incompletely penetrant feature of patients with Pallister-Hall syndrome caused by mutations of the GLI3 gene. This study demonstrated the role of GLI3 in sonic hedgehog signaling and how mutations affect this developmental pathway in the inner ear.